[The relationship between insulin resistance and risk of long-term mortality in people without diabetes: a 30-year follow-up of the Daqing Diabetes Study].

Objective: To determine whether insulin resistance is associated with all-cause mortality in subjects without diabetes. Methods: A total of 505 participants without diabetes, 198 with normal glucose tolerance (NGT) and 307 with impaired glucose tolerance (IGT), were recruited from the Daqing Diabetes Study. The participants were followed up for 30 years. They were stratified into three groups (tertiles) according to baseline homeostasis model assessment of insulin resistance(HOMA-IR) levels, as the HOMA-IR 0, the HOMA-IR 1 and the HOMA-IR 2 groups, to assess the predictive effect of insulin resistance on risk of all-cause mortality. Results: During the 30-year follow-up, 52, 56 and 78 participants died across the three HOMA-IR groups, respectively. The corresponding mortality per 1 000 person-years (95%CI) were 12.12 (9.56-15.01), 13.10 (10.46-16.03) and 19.91 (16.73-23.15), respectively. Participants in the HOMA-IR 2 group had a significantly higher risk of death than those in the HOMA-IR 0 group after adjustment of age, sex and smoking status (HR=1.97,95%CI 1.38-2.81, P<0.001). Cox analyses showed that a one standard deviation increase in HOMA-IR was associated with a 22% increase in the mortality after adjustment of potential confounders (HR=1.22, 95%CI 1.08-1.39, P=0.002). Conclusions: Insulin resistance is associated with increased risk of all-cause death in Chinese people without diabetes, suggesting that improving insulin resistance could be beneficial for people without diabetic in reducing risk of long-term all-cause mortality.

[Association between physical activity and risk of stroke among adults aged 40 years and above: a prospective cohort study].

Objective: To examine the effect of physical activity (PA) on the incident risk of stroke among adults aged 40 years and above. Methods: The baseline data including PA and demographic characteristics were obtained from the Adult Chronic Disease Surveillance with population representativeness in Ningbo in 2015. The follow-up data of interested health outcomes from 2015 to 2019 were retrieved from a population-based Integrated Noncommunicable Disease Collaborative Management System in Ningbo. The two databases were matched to form a queue. PA was divided into three levels of low-intensity, moderate-intensity, and vigorous-intensity according to the metabolic equivalents (METs) spent per week. Cox regression model was used to calculate the hazard ratio (HR) and 95% confidence interval. Results: A total of 3 353 subjects were included at baseline survey in 2015. Until Dec 31, 2019, there had been 31 stroke events had occurred since then, with accumulative incidence rate of 242/100 000, and an average follow-up time of (50.28±2.54) months. When adjusted for gender, age, education level, smoking status, alcohol consumption, BMI and hypertension, multivariate Cox regression analysis showed that greater PA was associated with a 37.9% reduction of incidence of stroke (HR=0.621,95%CI:0.393-0.983). Compared with those who had low-intensity PA, those who were with vigorous-intensity. PA appeared associated with a 63.1% decrease in the incidence of stroke (HR=0.369, 95%CI: 0.139-0.976). However, there was no statistical significance with moderate-intensity PA (HR=0.712,95%CI:0.323-1.569), noticed. Conclusions: Greater PA is likely to reduce the incidence of stroke. Our findings indicated that people should be encouraged to increase the PA level and developing a healthy supportive environment in the community.

Progesterone withdrawal increases the anxiolytic actions of gaboxadol: role of alpha4betadelta GABA(A) receptors.

Hippocampal alpha4betadelta GABA(A) receptors (GABA(A)-R) are increased following progesterone withdrawal (PWD) in a rodent model of premenstrual anxiety. This alpha4betadelta receptor isoform uniquely responds to the GABA agonist gaboxadol (THIP) with a maximum current greater than that gated by GABA, and is potentiated more by pentobarbital than are other GABA(A)-R. We therefore investigated the anxiolytic effects of these drugs using the elevated plus maze. Gaboxadol (1.25 mg/kg) was markedly more anxiolytic in animals undergoing PWD than in controls. Pentobarbital (10 mg/kg) also produced a greater anxiolytic effect during PWD. These results suggest that the pharmacological properties of alpha4betadelta GABA(A)-R following PWD are evident behaviorally. Alterations in the alpha4betadelta GABA(A)-R population may have implications for the etiology and treatment of premenstrual syndrome.

Progesterone withdrawal increases the alpha4 subunit of the GABA(A) receptor in male rats in association with anxiety and altered pharmacology - a comparison with female rats.

Withdrawal from the neurosteroid 3alpha,5alpha-allopregnanolone after chronic administration of progesterone increases anxiety in female rats and up-regulates the alpha4 subunit of the GABA(A) receptor (GABA(A)-R) in the hippocampus. We investigated if these phenomena would also occur in male rats. Progesterone withdrawal (PWD) induced higher alpha4 subunit expression in the hippocampus of both male and female rats, in association with increased anxiety (assessed in the elevated plus maze) comparable to effects previously reported. Because alpha4-containing GABA(A)-R are insensitive to the benzodiazepine (BDZ) lorazepam (LZM), and are positively modulated by flumazenil (FLU, a BDZ antagonist), we therefore tested the effects of these compounds following PWD. Using whole-cell patch clamp techniques, LZM-potentiation of GABA ((EC20))-gated current was markedly reduced in CA1 pyramidal cells of male rats undergoing PWD compared to controls, whereas FLU had no effect on GABA-gated current in control animals but increased it in PWD animals. Behaviorally, both male and female rats were significantly less sensitive to the anxiolytic effects of LZM. In contrast, FLU demonstrated significant anxiolytic effects following PWD. These data suggest that neurosteroid regulation of the alpha4 GABA(A)-R subunit may be a relevant mechanism underlying anxiety disorders, and that this phenomenon is not sex-specific.

Short-term exposure to a neuroactive steroid increases alpha4 GABA(A) receptor subunit levels in association with increased anxiety in the female rat.

Previous work from this laboratory has demonstrated that withdrawal from the neuroactive steroid 3alpha,5alpha-THP (3alpha-hydroxy-5alpha-pregnan-20-one) after 3-week exposure to its parent compound, progesterone (P), increases anxiety and produces benzodiazepine (BDZ) insensitivity in female rats. These events were linked to upregulation of the alpha4 subunit of the GABA(A) receptor (GABAR) in the hippocampus [Brain Res. 507 (1998) 91; Nature 392 (1998) 926; J. Neurosci. 18 (1998) 5275]. The present study investigates the role of shorter term hormone treatment on alpha4 subunit levels as well as relevant behavioral and pharmacological end-points related to GABAR function. After 2-3 days of P exposure, two- to threefold increases in alpha4 protein levels were observed, which declined to control values after 5-6 days of hormone exposure. This effect was due to the GABA-modulatory metabolite of P, 3alpha,5alpha-THP. alpha4 upregulation was inversely correlated with BDZ potentiation of GABA-gated current, assessed using whole cell patch clamp techniques on acutely isolated hippocampal pyramidal cells. A near total BDZ insensitivity was observed by 2-3 days of hormone exposure in association with the maximal increase in alpha4 levels. Up-regulation of the alpha4 GABAR subunit was also reflected by an increase in anxiety in the elevated plus maze. A significant decrease in open arm entries was observed after 72-h exposure to P, an effect which recovered by 6 days of P treatment. As demonstrated in vitro, alpha4 upregulation also resulted in a relative insensitivity to the anxiolytic actions of BDZ. These results suggest that short-term exposure to 3alpha,5alpha-THP produces changes in GABAR subunit composition similar to those that occur after chronic exposure and withdrawal from the steroid.

The phenobarbital response enhancer module in the human bilirubin UDP-glucuronosyltransferase UGT1A1 gene and regulation by the nuclear receptor CAR.

The UDP-glucuronosyltransferase, UGT1A1, is the critical enzyme responsible for detoxification of the potentially neurotoxic bilirubin by conjugating it with glucuronic acid. For decades, phenobarbital (PB) treatment for hyperbilirubinemia has been known to increase expression of the UGT1A1 gene in liver. We have now delineated the PB response activity to a 290-bp distal enhancer sequence (-3483/-3194) of the UGT1A1 gene. The enhancer contains 3 putative nuclear receptor motifs, and it was activated by the nuclear orphan receptor, human constitutive active receptor (hCAR), in cotransfected HepG2 cells. Bacterially expressed hCAR, acting as a heterodimer with in vitro-translated retinoid X receptor (RXRalpha), only bound to 1 of the 3 NR motifs, named gtNR1 in a gel-shift assay. Consistently, mutations of the gtNR1 site significantly decreased the activation by hCAR of the 290-bp DNA in transfection assays. Moreover, the 290-bp DNA was effectively activated in mouse primary hepatocytes in response to PB, offering an excellent clinical test for the examination of the responsiveness of the UGT1A1 to PB in the human population, particularly individuals with hyperbilirubinemia.

Withdrawal from 3alpha-OH-5alpha-pregnan-20-One using a pseudopregnancy model alters the kinetics of hippocampal GABAA-gated current and increases the GABAA receptor alpha4 subunit in association with increased anxiety.

In the present study, we have characterized properties of steroid withdrawal using a pseudopregnant rat model. This paradigm results in increased production of endogenous progesterone from ovarian sources and as such is a useful physiological model. "Withdrawal" from progesterone induced by ovariectomy on day 12 of pseudopregnancy resulted in increased anxiety, as determined by a decrease in open arm entries on the elevated plus maze compared to control rats and pseudopregnant animals not undergoing withdrawal. Similar findings were obtained 24 hr after administration of a 5alpha-reductase blocker to a pseudopregnant animal, suggesting that it is the GABAA-modulatory 3alpha-OH-5alpha-pregnan-20-one (3alpha, 5alpha-THP) that produces anxiogenic withdrawal symptoms. Twenty-four hours after steroid withdrawal, the time constant for decay of GABAA-gated current was also reduced sixfold, assessed using whole- cell patch-clamp procedures on pyramidal neurons acutely dissociated from CA1 hippocampus. Thus, 3alpha,5alpha-THP withdrawal results in a marked decrease in total GABAA current, a possible mechanism for its anxiogenic, proconvulsant sequelae. In addition, 3alpha,5alpha-THP withdrawal resulted in insensitivity to the normally potentiating effect of the benzodiazepine lorazepam (LZM) on GABAA-gated Cl- current. This withdrawal profile is similar to that reported for other GABAA-modulatory drugs such as the benzodiazepines (BDZs), barbiturates, and ethanol. These changes were also associated with significant two and threefold increases in both the mRNA and protein for the alpha4 subunit of the GABAA receptor, respectively, in hippocampus. The pseudopregnancy paradigm may be a useful model for periods of endogenous 3alpha,5alpha-THP withdrawal such as premenstrual syndrome and postpartum or postmenopausal dysphoria, when increased emotional lability and BDZ insensitivity have been reported.

GABA(A) receptor alpha4 subunit suppression prevents withdrawal properties of an endogenous steroid.

The hormone progesterone is readily converted to 3alpha-OH-5alpha-pregnan-20-one (3alpha,5alpha-THP) in the brains of males and females. In the brain, 3alpha,5alpha-THP acts like a sedative, decreasing anxiety and reducing seizure activity, by enhancing the function of GABA (gamma-aminobutyric acid), the brain's major inhibitory neurotransmitter. Symptoms of premenstrual syndrome (PMS), such as anxiety and seizure susceptibility, are associated with sharp declines in circulating levels of progesterone and, consequently, of levels of 3alpha,5alpha-THP in the brain. Abrupt discontinuation of use of sedatives such as benzodiazepines and ethanol can also produce PMS-like withdrawal symptoms. Here we report a progesterone-withdrawal paradigm, designed to mimic PMS and post-partum syndrome in a rat model. In this model, withdrawal of progesterone leads to increased seizure susceptibility and insensitivity to benzodiazepine sedatives through an effect on gene transcription. Specifically, this effect was due to reduced levels of 3alpha,5alpha-THP which enhance transcription of the gene encoding the alpha4 subunit of the GABA(A) receptor. We also find that increased susceptibility to seizure after progesferone withdrawal is due to a sixfold decrease in the decay time for GABA currents and consequent decreased inhibitory function. Blockade of the alpha4 gene transcript prevents these withdrawal properties. PMS symptoms may therefore be attributable, in part, to alterations in expression of GABA(A) receptor subunits as a result of progesterone withdrawal.

Similarities and differences in mechanisms of cardiotoxins, melittin and other myotoxins.

Myonecrosis induced in vivo by cardiotoxin, melittin, and Asp49 and Lys49 phospholipase A2 (PLA2) myotoxins involves rapid lysis of the sarcolemma, myofibril clumping, and hypercontraction of sarcomeres. In contrast, skeletal muscle necrosis induced by crotamine and myotoxin a is much slower, consisting of mitochondrial and sarcoplasmic reticulum swelling, myofibril degeneration, and lack of sarcolemma or transverse tubule damage. The mechanisms contributing to the myonecrosis induced by these peptides were evaluated. Two cardiotoxins and two Lys49 PLA2 myotoxins lysed primary cultures of human skeletal muscle within 24 hr at a concentration of 0.25 microM, while melittin, crotamine, and myotoxin a, and an Asp49 PLA2 myotoxin were non-cytolytic at concentrations up to 5.0 microM, suggesting that cytolysis is not a good measure of myotoxicity. Crotamine and the Lys49 PLA2 myotoxin altered Ca2+ ion flux in human heavy sarcoplasmic reticulum by opening the ryanocine receptor. Whole-cell patch-clamp studies demonstrated that administrating crotamine intracellularly increased Na+ currents. Free fatty acids, liberated by activation of tissue phospholipase C or by the PLA2 activity of the myotoxins, were monitored for crotamine, myotoxin a and a Lys49 PLA2 myotoxin in cell cultures in which the lipids had been radiolabeled. Only the Lys49 myotoxin produced significant amounts of fatty acid in cell cultures, supporting a potential role for fatty acid production only in the mechanism of sarcolemma-destroying myotoxins. These findings, coupled with those in the literature, support a hypothesis in which the myotoxins and/or products of lipase activity (e.g. fatty acids) are acting at a site existing on both the Na+ channel and a protein involved in Ca2+ release and probably serving a modulatory function for ion regulation. Based on the similarities in mechanisms between the toxins and fatty acids, the most likely site would be a fatty acid binding site on the protein (either similar to that on fatty acid binding proteins, or an acylated cysteine residue) or in the membrane.

Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5' hypersensitive site 2 of the beta-globin locus control region.

Much of our understanding of the process by which enhancers activate transcription has been gained from transient-transfection studies in which the DNA is not assembled with histones and other chromatin proteins as it is in the cell nucleus. To study the activation of a mammalian gene in a natural chromatin context in vivo, we constructed a minichromosome containing the human epsilon-globin gene and portions of the beta-globin locus control region (LCR). The minichromosomes replicate and are maintained at stable copy number in human erythroid cells. Expression of the minichromosomal epsilon-globin gene requires the presence of beta-globin LCR elements in cis, as is the case for the chromosomal gene. We determined the chromatin structure of the epsilon-globin gene in both the active and inactive states. The transcriptionally inactive locus is covered by an array of positioned nucleosomes extending over 1,400 bp. In minichromosomes with a (mu)LCR or DNase I-hypersensitive site 2 (HS2) which actively transcribe the epsilon-globin gene, the nucleosome at the promoter is altered or disrupted while positioning of nucleosomes in the rest of the locus is retained. All or virtually all minichromosomes are simultaneously hypersensitive to DNase I both at the promoter and at HS2. Transcriptional activation and promoter remodeling, as well as formation of the HS2 structure itself, depended on the presence of the NF-E2 binding motif in HS2. The nucleosome at the promoter which is altered upon activation is positioned over the transcriptional elements of the epsilon-globin gene, i.e., the TATA, CCAAT, and CACCC elements, and the GATA-1 site at -165. The simple availability of erythroid transcription factors that recognize these motifs is insufficient to allow expression. As in the chromosomal globin locus, regulation also occurs at the level of chromatin structure. These observations are consistent with the idea that one role of the beta-globin LCR is to maintain promoters free of nucleosomes. The restricted structural change observed upon transcriptional activation may indicate that the LCR need only make a specific contact with the proximal gene promoter to activate transcription.

Altered sodium current response to intracellular fatty acids in halothane-hypersensitive skeletal muscle.

Biopsies of human skeletal muscle were analyzed by an in vitro contracture test (IVCT) for responsiveness to a halothane challenge: noncontracting (nonresponsive; IVCT-) and contracting (IVCT+). A muscle biopsy that is IVCT+ indicates potential malignant hyperthermia (MH) susceptibility. Primary cultures were grown from portions of the skeletal muscle biopsies, and voltage-activated currents were measured by whole cell recording in the presence or absence of 2-5 microM intracellular arachidonic or oleic acids. In untreated IVCT- cells, Na+ currents were predominantly tetrodotoxin (TTX) insensitive, indicating that most of the current was carried through the embryonic SkM2 isoform of the Na+ channel. Inclusion of fatty acids in the recording pipette of IVCT- cells produced an increase in voltage-activated Na+ currents during 20 min of recording. Approximately 70% of currents in fatty acid-treated cells were TTX sensitive, indicating activation of the adult SkM1 isoform of the Na+ channel. In contrast to IVCT- cells, IVCT+ cells expressed Na+ currents that were predominantly TTX sensitive even in the absence of added fatty acid, thus showing a relatively large baseline functional expression of SkM1 channels. Addition of fatty acids to the recording pipette produced little further change in the magnitude or TTX sensitivity of the whole cell currents in IVCT+ cells, suggesting altered functional regulation of Na+ channels in MH muscle.

A lineage-specific Ca(2+)-activated K+ conductance in HL-60 cells.

Cells of the human promyelocytic cell line HL-60 can be controllably induced to terminally differentiate into either granulocytes or monocyte/macrophages. HL-60 promyelocytes and terminally differentiated macrophages express a K(+)-selective ion channel which is activated by intracellular free Ca2+ concentrations above 10(-7) M. Because of its voltage independence, this channel can be distinguished from the voltage- and Ca(2+)-activated family of outward-rectifying channels. The channel is selective for K+ against Na+ and is blocked by Ba2+, thus it may be similar to the Ca(2+)-activated K+ channel previously described in human macrophages. In its sensitivity to block by charybdotoxin, this channel also resembles a Ca(2+)-activated K+ channel of lymphocytes, which plays a role in activation-dependent hyperpolarization. In contrast to promyelocytes and macrophages, functional expression of the Ca(2+)-activated K+ channel is suppressed to nearly undetectable levels in granulocytes derived from HL-60 cells by retinoic acid-induced differentiation. These data suggest that signals which produce elevation of intracellular Ca2+ will hyperpolarize promyelocytes and differentiated macrophages by activating this conductance; however, signals which elevate free Ca2+ in granulocytes must act on other effectors, which may produce a different final influence on membrane potential.

Ca(2+)-insensitive modulation of a K+ conductance by inositol polyphosphates.

Macrophages derived from phorbol ester-induced human leukemic (HL-60) cells exhibit a voltage-activated inward rectifying potassium conductance which was modulated by macrophage colony-stimulating factor (Wieland, S. J., Chou, R. H., and Gong, Q. H. (1990) J. Cell. Physiol. 142, 643-651). Roles of intracellular messengers in this regulatory mechanism were investigated. Intracellular dialysis with inositol 1,3,4,5-tetrakisphosphate (IP4) or inositol 1,4,5-trisphosphate during tight-seal whole cell recording produced a rapid increase in the inward rectifying conductance. Changes in intracellular Ca2+ levels alone did not reproduce the stimulatory effect of these modulators. Intracellular dialysis with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) resulted in profound inhibition of this conductance. These data suggest a novel cellular function for inositol polyphosphates, particularly IP4, and show antagonistic modulation with GTP gamma S on a human macrophage inward rectifier.

Snake venom cardiotoxins and bee venom melittin activate phospholipase C activity in primary cultures of skeletal muscle.

The effects of cardiotoxin fractions from Naja naja kaouthia and Naja naja atra snake venoms and synthetic melittin peptide were examined on lipolytic activity in red blood cells and primary skeletal muscle cultures. Both native cardiotoxin fractions caused considerable production of free fatty acids in red blood cells. This production was abolished when the fractions were first treated with p-bromophenacyl bromide to reduce the venom phospholipase A2 activity contamination. In equine and human primary cultures of skeletal muscle, the N. n. kaouthia cardiotoxin (10 microM) and melittin (2 microM) caused a breakdown of phospholipids and production of free fatty acids and diacylglycerol in the absence of lysophospholipid formation. Additionally, melittin at higher concentrations (10 microM) caused triglyceride breakdown. These studies do not support the suggestion that snake venom cardiotoxins and melittin selectively activate endogenous phospholipase A2 activity. Instead, the toxins primarily activate endogenous phospholipase C activity and, in the case of melittin at high concentrations, triglyceride lipase activity.

Effects of a cardiotoxin from Naja naja kaouthia venom on skeletal muscle: involvement of calcium-induced calcium release, sodium ion currents and phospholipases A2 and C.

Snake venom cardiotoxin (CTX) fractions induce contractures of skeletal muscle and hemolysis of red blood cells. The fractions also contain trace amounts of venom-derived phospholipase A2 (PLA2) contamination and activate tissue phospholipase C (PLC) activity. The present study examines the mechanisms of action of a CTX fraction from Naja naja kaouthia venom in skeletal muscle. Sphingosine competitively antagonized CTX-induced red blood cell hemolysis, but not skeletal muscle contractures. CTX rapidly lowered the threshold for Ca(2+)-induced Ca2+ release in heavy sarcoplasmic reticulum fractions, as monitored with arsenazo III. There was also a slower time-dependent reduction of Na+ currents, as assessed by whole cell patch-clamp techniques. The CTX fractions elevated levels of free fatty acids and diacylglycerol for 2 hr in primary cultures of human skeletal muscle by a combined action of venom-derived PLA2 contamination in the fraction and activation of endogenous PLC activity. The activation of tissue PLC activity could be readily distinguished from the contribution of the venom PLA2 by p-bromophenacyl bromide treatment of CTX fractions. The mechanism of action involved in contractures of skeletal muscle appears to be related to the immediate and specific effect of CTX (Ca2+ release by the sarcoplasmic reticulum), while the mechanisms involved in hemolysis of red blood cells and decreased Na+ currents in skeletal muscle most likely relate to long-term effects on lipid metabolism.

Macrophage-colony-stimulating factor (CSF-1) modulates a differentiation-specific inward-rectifying potassium current in human leukemic (HL-60) cells.

A voltage-activated inward-rectifying K+ conductance (lKi) appears in human promyelocytic leukemia (HL-60) cells during phorbol ester-induced differentiation into macrophages. This conductance was detected in the cells 24 hours after exposure to phorbol-12-myristate-13-acetate (PMA), as the cells began to express the macrophage phenotype, and continued to increase for 4 days after PMA exposure. The magnitude of inward current was a function of external K+; current was blocked by extracellular or intracellular Cs+ and by extracellular Ba++. Hyperpolarization produced activation at membrane potentials more negative than -80 mV, and a slower, partial inactivation also occurred at potentials more negative than -100 mV. This conductance was not detected in proliferating cells nor in granulocytes derived from HL-60 cells which were induced to differentiate with retinoic acid (RA). Exposure of differentiated macrophages to recombinant human CSF-1 produced inhibition of the lKi beginning within 1 minute after exposure. CSF-1 inhibition of lKi channels in cell-attached patches indicated that channel modulation was via intracellular mediators. The rapid inhibition of the inward rectifier by the macrophage-specific CSF-1 appears to be one of the earliest cellular responses to this factor.

Malignant hyperthermia: slow sodium current in cultured human muscle cells.

Voltage-activated ion currents were measured in cultured skeletal muscle myoballs. Cultures were generated from biopsies from patients referred for diagnosis of susceptibility to malignant hyperthermia (MH); diagnosis of susceptibility (MH+) or nonsusceptibility (MH-) was made on the basis of in vitro halothane-induced contracture of a separate piece of biopsy. Measurements of ion currents were made at room temperature in the absence of anesthetic agents, using tight-seal whole-cell recording. Fast transient Na+ currents and delayed outward K+ currents were similar in magnitude and kinetics in cells from MH+ and MH- patients. An additional slowly inactivating inward current component was commonly observed in cells from MH+ patients. This current was blockable by tetrodotoxin and was carried by Na+ but not by Ba2+. The component was less frequently observed and was of a lower magnitude in cells from MH- patients. The increased magnitude of the slow inward current observed in cultured muscle cells from MH+ patients may be a manifestation of the lesion that causes MH.

Effect of a phospholipase A2 with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents.

The action of a 16,300 mol. wt phospholipase A2 with cardiotoxin-like properties from Bungarus fasciatus venom on membrane electrical properties of two human cell types was examined in vitro by using tight-seal whole-cell recording methods. Epithelial cells exhibited a voltage- and Ca2(+)-activated K+ current; the sensitivity for voltage activation of the K+ current was enhanced by increasing free Ca2+ in the recording pipette from 10(-8) M to 2 x 10(-6) M. In contrast, peripheral blood lymphocytes possessed voltage-activated K+ currents that were inhibited by increasing intracellular Ca2+. Exposure of either preparation to B. fasciatus toxin (0.2-5 x 10(-6) M) for up to 30 min in the bath did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of -140 mV to -40 mV. However, the sensitivity for voltage activation of the K+ current was enhanced in the epithelial cells even at the lowest concentrations tested. In contrast to the results with epithelial cells, toxin exposure inhibited the activation of voltage-activated K- currents in human lymphocytes, suggesting a specific increase in intracellular Ca2- levels in both cell types. The fluorescent probe indo-1/AM was used to monitor cytoplasmic Ca2+ levels. Exposure of either lymphocytes or epithelial cells to toxin (10(-6) M) resulted in a transient increase in Ca2+. However, while the Ca2+ response to toxin was transient, K-channel modulation by the toxin appeared to be irreversible over the experimental time course. The longer-lasting modulation of Ca2(+)-regulated K+ channels may reflect an irreversible action of the B. fasciatus phospholipase A2 on a Ca2+-dependent regulatory process.